A key feature of clinically effective human anti-tumor immunity is the ability to raise T cell responses that are strong and exquisitely tumor-specific and, to overcome immune escape and tumor heterogeneity, simultaneously target multiple antigens. Despite the critical importance of the tumor-targeted, antigen-specific T cell, the enormous heterogeneity of T cell populations and the complexity of the T cell receptor (TCR) locus have presented technical challenges to the systematic characterization of antigen-specific T cell immunity. In particular, only limited tools are available to evaluate the key molecular signatures of this cell - the specific TCR molecule (which requires paired TCR? and TCR? chains for epitope recognition) and the phenotype defining pattern of immune pathway-related expressed genes - at the individual cell level rather than the broad population level. The Broad Technology Laboratories (BTL) have recently developed an innovative single cell bar-coded droplet-based (SCBD) sequencing technology that dramatically expands the number of T cells that can be individually examined for TCR gene identification (as well as for other immune markers). This engineered droplet emulsion approach is uniquely equipped to meet the challenge of the highly diverse T cell repertoire. Building on studies from the parent grant (1RO1 CA 155010-03) that have pioneered the characterization of personal tumor neoantigens, the proposed grant will take a critical and complementary step forward by characterizing in detail the T cell population responding to neoantigens. Aim 1 will unlock the potential of this technology by demonstrating that the variable sequences of paired TCR? and TCR? chains targeting a known antigen can be reliably and accurately identified from complex T cell mixtures. Aim 2 will show that the technology can be used to identify specific TCR pairs and their molecular targets from patients that have been vaccinated with a complex immunogen (whole irradiated tumor cells). The experimental approach described here is more reliable than previous methods as it does not depend on variable T cell culture techniques, and is more unbiased because it does not depend on specific predictions of peptides binding to specific HLAs. Importantly, this approach uses small numbers of input T cells and thus can be used for precious patient samples. These proof-of-concept studies are anticipated to lay the foundation for developing a path for systemically evaluating, in conjunction with next-generation DNA and RNA sequencing of tumor samples, the antigen-specificity of T cell responses of patients from vaccination and other immunotherapy trials. This platform is anticipated to be generalizable across any human disease setting in which TCR repertoire and antigen specificity information is needed.